Physiological analysis of
lactobacillus rhamnosus VTT
E-97800
Adaptive response to osmotic stress induced
by trehalose
E.O. Sunny-Roberts and D. Knorr
Department of Food Biotechnology and Process Engineering,
Berlin University of Technology, Berlin, Germany
Abstract
Purpose – This paper aims to describe the physiological analysis of L. rhamnosus VTT E-97800 and
its adaptive response to osmotic stress induced by trehalose.
Design/methodology/approach – Cells of L. rhamnosus E800 in the stationary phase of growth
were subjected to osmotic stress induced by trehalose treatments. The effects of osmotic stress on the
viability of the study strain were determined by conducting flow cytometric analysis with
carboxyfluorescein diacetate (cFDA) and propidium iodide (PI) and by observing the corresponding
cells growth on MRS agar plates. Osmotic-induced changes of esterase activity and membrane
integrity were monitored. Ability to extrude intracellular accumulated cF (additional vitality marker)
was taken into consideration.
Findings – The fluorescence-based approach gave additional insights on osmotic induced changes of
cellular events, which could not be explicitly assessed by culture techniques. Trehalose treatments
caused a transient membrane permeabilization as revealed by a gradual decrease in esterase activity (a
measure of enzyme activity and thus of viability) with increase in trehalose molarity. However,
culturability on MRS agar was not significantly affected. Membrane integrity was maintained and
there was an improvement in the ability of cells to extrude intracellular accumulated cF.
Originality/value – The paper provides a comparative study of the conventional culture techniques
and the flow cytometric viability assessment which showed that esterase activity cannot be relied on
to ascertain the culturability and viability status of an organism.
Keywords Food preservation, Metabolic diseases, Micro-organisms, Biotechnology
Paper type Research paper
Introduction
The probiotic strain Lactobacillus rhamnosus E-97800 (E800), isolated from human
intestines with lactitol enrichment has been characterized and identified by molecular
biological typing methods RAPD, automatic ribotyping and PFGE. The probiotic
properties such as good tolerance to low pH, bile pancreatic juice, and the ability to
adhere to both the intestinal epithelium cell line Caco-2 cells and human ileostomy
glycoproteins have been reported (Tynkkynen et al., 1999). Technologically, it adheres
well to fibres creating a possibility for cereal-based probiotic products.
Bacteria have evolved stress-sensing systems and defences against stress, which
allow them to withstand harsh conditions and sudden environmental changes (Van de
The current issue and full text archive of this journal is available at
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The authors are grateful to the German Academic Exchange Programme for its financial support
in the course of this work. Many thanks go to Irene Hemmerich for her technical assistance.
Lactobacillus
rhamnosus VTT
E-97800
735
British Food Journal
Vol. 109 No. 9, 2007
pp. 735-748
qEmerald Group Publishing Limited
0007-070X
DOI 10.1108/00070700710780706
Guchte et al., 2002). These stress responses are characterized by the transient induction
of general and specific proteins and by physiological changes that generally enhance
an organism’s ability to withstand more adverse environmental conditions (Ang et al.,
1991). In the case of osmotic stress, the significant physiological changes reported in
bacteria include the induction of stress proteins as well as intracellular accumulation of
compatible solutes (Clark and Parker, 1984; Kets and de Bont, 1994; Kets et al., 1994;
Welsh and Herbert, 1999; Prasad et al., 2003), for example, trehalose, amino acids,
sucrose, mannitol etc. These solutes restore turgor pressure and membrane tension to
low levels similar to those that occur before osmotic shift (Csonka and Hansen, 1999),
preserve enzyme activity and protein stability, and maintain the integrity and stability
of membranes and nucleic acids (Brown, 1990; Liu et al., 1998).
Trehalose is a disaccharide that is ubiquitous in the biosphere. It consists of two
sub-units of glucose bound by an
a
:!1 linkage (a-D-glucopyranosil
a-D-glucopyranoside) and is thus non-reducing. Trehalose has been isolated and
characterized from a large variety of both prokaryotic and eukaryotic organisms,
ranging from bacteria and plants to mammals (Argu
¨elles, 2000). It possesses several
unique physical properties, which include nonhygroscopic glass formation and the
absence of internal hydrogen bond formation. These features account for the principal
role of trehalose as a stress metabolite.
Among the ways to preserve food products, increased osmotic pressure, i.e.
lowering of water activity (a
w
) is one of the most widely used. Desiccation or addition
of high amounts of osmotically active compounds such as salts and sugars lowers the
water activity of the food. Most of these compatible solutes are present in significant
amounts in foods, thus allowing growth at reduced water activities.
The potential role that trehalose could have as a biotechnological tool include
serving as a key element in food preparations subject to drying processes or
concentration (Schiraldi et al., 2002). In these cases, the incorporation of probiotics into
such foods is practised for consumers’ health benefit purposes.
Flow cytometry is a rapid and sensitive method that can be performed by double
staining of cells with carboxyfluorescein diacetate (cFDA) and propidium iodide (PI).
CFDA is used for the evaluation of cellular enzymatic activity. It is a lipophilic,
non-fluorescent precursor that readily diffuses across the cell membrane.
Intracellularly, unspecific esterase hydrolyses the diacetate group into a polar,
membrane impermeant fluorescent compound, carboxyfluorescein (cF). For cells to be
regarded as viable, this probe requires both active intracellular enzymes and intact
membranes (Hoefel et al., 2003). PI is a membrane-impermeant, nucleotide-binding
probe, which cannot penetrate cells with intact membranes, but once membrane
integrity is lost, it diffuses into and stains the nucleic acids.
In this paper, the induction of osmotic stress by the presence of trehalose in a
complex rich medium, on a model probiotic bacterium is reported. The study was
approached by measuring the fluorescence-related parameters and analysing the plate
counts; then making a comparative analysis between both.
Materials and methods
Bacterial strain and growth conditions
The strain of Lactobacillus rhamnosus E800 used was obtained from VTT culture
collection (Espoo, Finland). The culture which was sent in freeze-dried form in glass
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736
ampoule was later stored as glass beads cultures (Roti
(R)
_Store, Carl-Roth, Karlsruhe,
D) in a 280
o
C freezer (U101, New Brunswick Scientific, Nu
¨rtingen, D) for long-term
maintenance. One bead from deep-frozen culture was transferred into MRS broth
(Oxoid, Basingstoke, UK) and incubated over-night. This broth was later used to
inoculate a final broth (50 ml) at OD
600
0.1. Growth was carried out at 37
o
C over a
period of 24 h and monitored spectrophotometrically at 600 nm (Graphicord uV-240,
Schimadzu, JPN).
Media preparation
High osmolarity media were obtained by adding trehalose (0.1M, 0.3M, 0.4M, 0.6M,
0.9M, 1.2M and 1.5M) to the MRS broth (5 ml) (Oxford, Basingstoke, UK) at
concentrations indicated between the brackets. MRS broth without trehalose served as
control. The osmolarities of the solutions were measured (51308 Vapour Pressure
Osmometer Wescor Inc.).
Flowcytometry
Stress treatments. Stationary growth phase cultures were harvested, washed twice in
Ringer’s solution (No. 15525, Merck, Darmstadt, D) and finally resuspended in Ringer’s
solution to an OD
600
value of 10 (Ananta et al., 2004). This corresponded to a cell
concentration of 3.4x10
9
CFU.mL
-1
.
Equal volume of the cell concentrate was treated with equal volume of the
MRS-trehalose solution at 37
o
C for 30 mins. Cells treated with MRS broth only served
as controls. Heat-treated cells (95
o
C, 15 mins) served as negative controls. The cells
viability was then assessed by flow cytometric methods and plate counts enumeration.
Plate enumeration method
Treated samples and control samples were serially diluted in Ringer’s solution
(No. 15525, Merck, Darmstadt, D) and cultured on MRS agar plate by drop count
technique (Miles and Misra, 1938). The viable cell numbers were determined after 48 h
of incubation at 37
o
C under anaerobic conditions produced by anaerobic kits
(AnaerocultwA, Merck, Darmstadt, D).
The impact of osmotic treatment on cell viability, as assessed by plate count method
was expressed as the logarithmic value of relative survivor fraction (logN/N
o
). N refers
to the bacterial count following osmotic exposure, while N
o
refers to the initial count
before osmotic exposure.
Esterase activity and membrane integrity
Cells were incubated with 50 mm cFDA (Molecular Probes, Inc., Leiden, NL) at 37
o
C for
10 mins. cFDA is an esterase substrate that yields the fluorescent carboxyfluorescein
(cF) upon hydrolysis. Cells were washed to remove excess cFDA and 30 mmPI
(Molecular Probes Inc., Leiden, NL) was added. Cells were incubated in ice bath for
10 mins to allow labelling of membrane-compromised cells (Ananta et al., 2004).
cF extrusion activity and kinetics of extrusion
cF stained cells were further incubated with 20mM glucose at 37
o
C for 40 mins in order
to measure the performance of cells in extruding intracellular accumulated cF (Bunthof
Lactobacillus
rhamnosus VTT
E-97800
737
et al., 1999). Kinetic measurements were performed by withdrawing samples at
intervals over a period of 40 mins to monitor release of cF from glucose energized cells.
Flow cytometric measurement
Analysis was performed on a CoulterwEPICSwXL_MCL flow cytometer (Beckman
Coulter Inc., Miami, FL, USA) equipped with a 15mW 488 nm air-cooled argon laser.
Cells were delivered at the low flow rate, corresponding to 400-600 events. Forward
scatter (FS), side scatter (SS), green (FL1) and red fluorescence (FL3) of each single cell
were measured, amplified and converted into digital signals for further analysis. CF
emits green fluorescence at 530 nm following excitation with laser light at 488 nm,
whereas red fluorescence at 635 nm is emitted by PI-stained cells.
A set of band pass filters of 525 nm (505-545 nm) and 620 nm (605-635 nm) was used
to collect green fluorescence (FL1) and red fluorescence (FL3) respectively. All
registered signals were logarithmically amplified. A gate created in the density-plot of
FS vs. SS was preset to discriminate bacteria from artefacts. Data were analysed with
the software package Expo32 ADC (Beckman-Coulter inc., Miami, FL, USA). All
detectors were calibrated with FlowCheck
TM
Flourospheres (Beckman-Coulter Inc.,
Miami, FL, USA).
Data analysis
Density plot analysis of FL1 vs. FL3 was conducted as described by Ananta et al.
(2004). Density plot was used to resolve the fluorescence properties of the population
measured by flow cytometer. The population was then differentiated and gated.
Residual esterase activity following osmotic treatment was calculated using
equation (1):
EAð%Þ¼ð#A4p=#A4ctrlÞx100 ð1Þ
Where EA is the residual enzymatic activity in response to a particular osmotic
treatment, (A4p is the percentage of population in (A4 following osmotic treatment,
(A4ctrl is the percentage of population in (A4 prior to osmotic treatment.
The performance of cells in extruding intracellular accumulated dye is calculated
using equation (2):
cFAð%Þ¼ð12#A4Glu=#A4Þx100 ð2Þ
Where cFA is the measure of performance in extruding cF, (A4
Glu
is the percentage of
population in (A4 following glucose addition and 40 mins incubation, (A4 is the
percentage of population in (A4 prior to glucose addition.
The kinetic of relative number of population extruding the intracellular
accumulated dye is calculated in equation (3) thus:
RcFð%Þ¼ð#A4t2Glu=#A4t¼0Þx100 ð3Þ
where RcF is the relative number of cells still stained with cF in (A4 following
glucose addition, (A4
t-Glu
is the percentage of cells still stained with cF in (A4
following glucose addition and incubation t min, (A4
t¼0
is the percentage of cells still
stained with cF in (A4 prior to glucose addition.
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Statistical analysis
The correlation between the cell viability and osmotic-induced changes on the
physiology of LGG was tested by one-way ANOVA test. Differences were considered
significant at p(0.05 level of probability. This was performed with Origin7 software
package (Origin Lab, Northampton, MA, USA).
Results and discussion
The osmotic strength of MRS broth before trehalose addition was 365mOs. Kg
21
H
2
0.
The incorporation of trehalose subsequently generated higher osmotic conditions
(Figure 1). The osmolarity of MRS medium containing 1.5M trehalose was greater than
3000 mOs. Kg
21
H
2
0. The influence of these conditions on the viability of Lactobacillus
rhamnosus E800 was tested, since one of the alternative methods of improving the
viability of probiotic cultures is the utilization of their stress adaptation.
Intracellular esterase activity and membrane integrity
Bacterial populations were differentiated into four quadrants (Figures 2, 3 and 4).
Populations in: No. 1 have their membranes compromised and they do not possess
esterase activity; No. 2 have their membranes minimally damaged and possess esterase
activity; No. 3 do not possess esterase activity, membranes are intact; and No. 4 possess
esterase activity and their membranes are intact.
Cells possessed residual esterase activity, which reduced with increase in sugar
molarity (Figure 5). FL1-FL3 density plots showed the existence of greater parts of cells
in No. 4. These cells had intact membranes and possessed esterase activity but some of
these moved to No. 3 (Figure 2). The presence of such populations in No. 3 could be
taken to indicate a loss of esterase activity. A further study was undertaken, and it was
observed that membranes were permeabilized thus allowing intracellular accumulated
cF, molecular weight 376, to leak. At physiological pH, cF has predominantly a
three-fold negative charge thus making it practically impermeable. The inability of PI
(molecular weight 668) to penetrate through the membrane into the cells, as a result of
low degree of permeabilization, signifies the ability of the cells to maintain their
Figure 1.
Graphical representation
of the osmolarity of
prepared MRS-trehalose
solutions
Lactobacillus
rhamnosus VTT
E-97800
739
Figure 2.
Flow cytometry density
plots of FL1 vs. FL3 of
Lactobacillus rhamnosus
E800 for evaluating the
impact of incubation in
trehalose solution at
different molarities on
their membrane integrity
and cF accumulation
capacity
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109,9
740
Figure 3.
Flow cytometry density
plots of FL1 vs. FL3 of
Lactobacillus rhamnosus
E800 to assess the impact
of incubation in trehalose
at different molarities on
their cF-extrusion activity
Lactobacillus
rhamnosus VTT
E-97800
741
Figure 4.
Flow cytometric
assessment of the kinetics
of cF efflux upon
energizing with glucose as
shown by density plots
flourescent pattern (FL1
vs. FL3) of the untreated
(upper row) and 0.6M
trehalose treated (lower
row) populations
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109,9
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membrane integrity. Bunthof et al. (1999) observed a gradual decrease of intracellular
cF in cF-labelled non-energized Lactobacillus lactis cells.
Leslie et al. (1995) observed a higher internal trehalose concentration when bacterial
cells were resuspended in trehalose at temperatures below the start of the phase
transition than those resuspended at 20
o
C and above. They reported that when cells
start to enter their phase transition, the membranes become leaky and sugar flows
down its concentration gradient into the cells. Thus, the permeabilization of the
membranes of the study strain could have contributed to the accumulation of trehalose
inside its cells.
The occurrence of esterase activity (Figure 5) was not in correlation with
culturability recorded on MRS agar plates (Figure 6), indicating that membrane
permeabilization was transient. Lactobacillus rhamnosus E800 must have made use of
repair mechanism during cultivation on MRS agar. The intracellular accumulation of
cF and the occurrence of esterase activity do not necessarily reflect crucial metabolic
activities which are involved in the maintenance of reproductive growth (Vives-Rego
et al., 2000).
Extrusion of intracellular accumulated dye
The ability of L. rhamnosus E800 to extrude accumulated cF upon glucose energizing
was investigated. This was conducted as an additional measure of cell viability to give
more information about physiological condition of the cells.
FL1-FL3 density plot analysis showed the extrusion of intracellular cF upon
energizing with 20mM glucose as represented by the shift of population in #4 to #3
(Figure 3) due to florescence loss. There was no significant record of the perturbation of
the extrusion activity (Figure 7). Cells that exhibited this additional measure of
viability were also culturable.
Some bacteria were reported to complete extrusion of accumulated dye in 20 min
(Bunthof et al., 1999; Ananta et al., 2004), contrary to a period of 40 min taken by
Figure 5.
Impact of trehalose
treatment at 37
o
C for 30
mins on esterase activity
at different molarities as
derived from fluorescent
density plots in Figure 2
Lactobacillus
rhamnosus VTT
E-97800
743
L. rhamnosus E800 to completely extrude the dye. Although the mentioned authors
prepared cells suspensions in buffer while Ringers solution was used in the present
study nevertheless, the duration of extrusion could be strain-specific.
Trehalose improved cells’ pump activity, which was most likely mediated by an
ATP-driven transport system. ATP production and rapid extrusion of cF upon
energizing were observed despite dissipation of proton motive force by addition of
Figure 6.
The impact of trehalose
treatment on the viability
of E800 assessed by plate
counting method and
exhibited as the
logarithmic value of
surviving cells (N/N
o
)
Figure 7.
Graphical representation
of cF-extrusion activity of
cells as affected by
trehalose treatments at
different molarities
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109,9
744
ionophores, valiomycin and nigericin (Molenaar et al., 1992; Bunthof et al., 1999). The
level of cF leakage due to the permeabilization of membranes was not as pronounced as
the quick extrusion observed upon energizing by glucose addition.
Kinetically, the rate of cF extrusion upon energizing was higher in 0.6M trehalose-
treated cells than in control cells, however both the treated and the untreated cells
completed extrusion at the end of incubation (Figures 4 and 8).
Trehalose was able to improve cF extrusion rate in Lactobacillus rhamnosus E800
therefore, this sugar can be used to enhance the resistance of probiotics to drugs or
toxic compounds, for example, antibiotics. Toxic compounds have always been part of
the natural habitat of micro-organisms but micro-organisms can reduce the
intracellular concentration of drugs by developing drug efflux systems that export
lipophilic drugs before these compounds have chance to find their cellular targets (Van
Veen and Konings, 1997; Van Veen et al., 1999). Increasing multidrug resistance have
been recorded in both gram-positive and gram-negative pathogens, which are
responsible for the most devastating and prevalent diseases of humans and domestic
animals (Borst and Ouellette, 1995). The use of trehalose in pharmaceuticals should be
limited to the manufacture of drugs that are not meant for antimicrobial actions.
Micro-organisms face a sudden change in environmental conditions and most have
been recognised to develop mechanisms of adaptation and survival. Under osmotic
conditions, they survive by accumulating compatible solutes. HPLC analysis of sugars
revealed the presence of trehalose in the cells and also in the stress medium of treated
cells only (data not shown in this report). The ability to accumulate trehalose is the
result of an elaborate genetic system, which is regulated by osmolarity (Argu
¨elles,
2000). The ability of cells to take up disaccharides so that they are present on both
Figure 8.
Graphical representation
of the relative numbers of
cF stained cells in the
presence of 20mM glucose
at increasing period of
incubation as derived from
the density plots in
Figure 4
Lactobacillus
rhamnosus VTT
E-97800
745
sides of the membrane and in contact with internal, cytosolic proteins increased the
tolerance of cells to preservation processes, e.g. drying.
Yeast extract, a component of MRS medium, contains betaine but this
osmoprotectant does not protect against non-electrolyte stress in lactic acid bacteria.
The uptake of sugar together with the accumulation of glycine betaine may eventually
result in the hyper-osmolarity of the cytoplasm, which will then be compensated by net
exit of glycine-betaine. This uptake most likely occurs by facilitated diffusion via a
system with a very low affinity for the substrates, which is consistent with the inability
of the sugars to serve as compatible solutes (at low substrate concentration) and may
be subject to osmotic regulation and transport system(s) may be more active under
hyper-osmotic conditions (Glaasker et al., 1998).
In a preliminary study (unpublished), it was observed that there was no significant
difference between the response of cells in the logarithm-phase of growth and the
stationary phase cells to osmotic stress by trehalose despite the fact that the latter are
known to develop a general stress resistance to various stresses. Stress responses may
be used to enhance the survival of probiotic bacteria in stressful conditions and to
improve their technological properties (Van de Gutche et al., 2002). The use of
stationary phase cells meets the need of the industrial production of cultures where
enough cells densities are needed before harvest.
Conclusions
In this study, the use of FCM and plate counts method for the viability assessment of
L. rhamnosus E800 subjected to sugar stress was investigated. Cell suspensions were
exposed to trehalose treatment at different molarities and fluorescent parameters were
compared with plate counts. Trehalose treated cells were vital, having cytoplasmic
membranes with selective permeability. Though there was a decrease in the cF
fluorescence labelled cells, the exclusion of PI showed that the integrity of membranes
was still maintained. Moreover, the experiments showed that the effects of osmotic
stress on esterase activity as a result of cF retention did not agree with culturability on
MRS agar plates. The esterase activity decreased in all treated samples but it never
became a limiting factor for cF extrusion and culturability. The data presented so far
showed that the viability of Lactobacillus rhamnosus E800 was not affected under the
tested hyper- osmotic conditions; therefore, the strain could be a good candidate in the
processing of foods containing trehalose. Moreover, any substance, which was
expected to increase the tolerance of organisms when dried, should be able to have
access into the cells in order to protect the proteins; and also to protect the membranes.
Trehalose meets these requirements.
References
Ananta, E., Heinz, V. and Knorr, D. (2004), “Assessment of high pressure induced damage on
Lactobacillus rhamnosus GG by flow cytometry”, Food Microbiology, Vol. 21, pp. 567-77.
Ang, D., Libereck, K., Skowyra, D., Zylicz, M. and Georgopoulous, C. (1991), “Biological role and
regulation of the universally conserved heat shock proteins”, Journal of Biological
Chemistry, Vol. 266, pp. 24233-6.
Argu
¨elles, J.C. (2000), “Physiological roles of trehalose in bacteria and yeasts: a comparative
analysis”, Archives of Microbiology, Vol. 174, pp. 217-24.
BFJ
109,9
746
Borst, P. and Ouellette, M. (1995), “New mechanisms of drug resistance in parasitic protozoa”,
Annual Review of Microbiology, Vol. 49, pp. 427-60.
Brown, A.D. (1990), Microbial Water Stress Physiology, John Wiley & Sons, Chichester, pp. 241-75.
Bunthof, C.J., Vanden Braak, S., Breeuwer, P., Rombouts, F.M. and Abee, T. (1999), “Rapid
fluorescence assessment of the viability of stressed Lactobacillus lactis”, Applied and
Environmental Microbiology, Vol. 65 No. 8, pp. 3681-9.
Clark, D. and Parker, J. (1984), “Proteins induced by high osmotic pressure Escherichia coli”,
FEMS Microbiology Letters, Vol. 25, pp. 81-3.
Csonka, L.N. and Hansen, A.D. (1999), “Prokaryotic osmoregulation: genetics and physiology”,
Annual Reviews of Microbiology, Vol. 45, pp. 569-606.
Glaasker, E., Tjan, F.S.B., Ter Steeg, P.F., Konings, W.N. and Poolman, B. (1998), “Physiological
response of lactobacillus plantarum to salt and nonelectrolyte stress”, Journal of
Bacteriology, Vol. 180 No. 17, pp. 4718-23.
Hoefel, D., Grooby, W.L., Moris, P.T., Andrew, S. and Saint, C.P. (2003), “A comparative study of
carboxyfluorescencein diacetate and carboxyfluorescein diacetate succimidyl ester as
indicators of bacterial activity”, Journal of Microbiological Methods, Vol. 53, pp. 379-88.
Kets, E.P.W. and de Bont, J.A.M. (1994), “Protective effect of betaine on survival of L. plantarum
subjected to dying”, FEMS Microbiology Letters, Vol. 116, pp. 251-6.
Kets, E.P.W., Galinski, E.A. and de Bont, J.A.M. (1994), “Carnitine: a novel compatible solute in
L. plantarum”, Archives of Microbiology, Vol. l62, pp. 243-8.
Leslie, S.B., Israeli, E.I., Light hart, B., Crowe, J.H. and Crowe, L.M. (1995), “Trehalose and sucrose
protect both membranes and proteins in intact bacteria during drying”, Applied and
Environmental Microbiology, Vol. 61 No. 10, pp. 3592-7.
Liu, Q., Asmundson, R.V., Gopal, P.K., Holland, R. and Crow, V.L. (1998), “Influence of reduced
water activity on lactose metabolism by Lactococcus lactis sub sp. cremoris at different pH
values”, Applied and Environmental Microbiology, Vol. 64 No. 6, pp. 2111-6.
Miles, A.A. and Misra, S.S. (1938), “The estimation of the bactericidal power of blood”, Journal of
Hygiene, Vol. 38, pp. 732-49.
Molenaar, D., Bolhuis, H., Abee, T., Poolman, B. and Konings, W.N. (1992), “The efflux of a
fluorescent probe is catalyzed by an ATP-driven extrusion system in Lactococcus lactis”,
Journal of Bacteriology, Vol. 174, pp. 3118-24.
Prasad, J., McJarrow, P. and Gopal, P. (2003), “Heat and osmotic stress responses of probiotic
Lactobacillus rhamnosus HN001 (DR20) in relaton to viability after drying”, Applied and
Environmental Microbiology, Vol. 69 No. 3, pp. 917-25.
Schiraldi, C., Di Lernia, I. and De Rosa, M. (2002), “Trehalose production: exploiting novel
approaches”, Trends in Biotechnology, Vol. 20 No. 10, pp. 420-5.
Tynkkynen, S., Satokari, R., Saarela, M., Mattila-Sandholm, T. and Sazelin, M. (1999),
“Comparison of ribotyping, randomly amplified polymorphic DNA analysis and
pulsed-field gel electrophoresis in typing of Lactobacillus rhamnosus and L. casei
strains”, Applied and Environmental Microbiology, Vol. 65 No. 9, pp. 3908-14.
Van de Gutche, M., Serror, P., Chervaux, C., Smokvina, T., Ehrlich, S.D. and Maunguin, E. (2002),
“Stress responses in lactic acid bacteria”, Antonie von Leeuwenhoek, Vol. 82, pp. 187-216.
Van Veen, H.W. and Konings, W.N. (1997), “Multidrug transporters from bacteria to man:
similarities in structure and function”, Cancer Biology, Vol. 8, pp. 183-91.
Lactobacillus
rhamnosus VTT
E-97800
747
Van Veen, H.W., Margolles, A., Putman, M., Sakamoto, K. and Konings, W.N. (1999), “Multidrug
resistance in lactic acid bacteria: molecular mechanisms and clinical relevance”, Antonie
van Leeuwenhoek, Vol. 76, pp. 347-52.
Vives-Rego, J., Lebaron, P. and Nebe-von Caron, G. (2000), “Current and future applications of
flow cytometry in aquatic microbiology”, FEMS Microbiology Reviews, Vol. 24, pp. 429-48.
Welsh, D.T. and Herbert, R.A. (1999), “Osmotically induced intracellular trehalose but not
glycine betaine accumulation promotes desiccation tolerance in Escherichia coli”, FEMS
Microbiology Letters, Vol. 174, pp. 57-63.
Corresponding author
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